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Primers, kit and detection method for detecting high-risk human papillomavirus E6/E7 mRNA

A technology of human papillomavirus and detection kit, which is applied in the field of life sciences, can solve problems such as time delay, complicated operation, and high cost, and achieve the effect of ensuring reliability, high sensitivity, and easy operation

Inactive Publication Date: 2018-08-03
HANGZHOU DIAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods can effectively detect high-risk HPV E6 / E7 mRNA in clinical samples, but the operation steps are cumbersome and costly, which is not conducive to the large-scale development of high-risk HPV screening.
[0007] At present, single-plex RT-PCR is the most widely used virus molecular biology detection method, which has the advantages of sensitivity, rapidity, and strong specificity. However, one reaction of single-plex RT-PCR can only detect one virus. If the reaction template contains two For more than one pathogen, multiple PCRs are required, which is not only complicated to operate, but also expensive and time-consuming

Method used

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  • Primers, kit and detection method for detecting high-risk human papillomavirus E6/E7 mRNA
  • Primers, kit and detection method for detecting high-risk human papillomavirus E6/E7 mRNA
  • Primers, kit and detection method for detecting high-risk human papillomavirus E6/E7 mRNA

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Embodiment 1

[0055] Embodiment one: the composition and main components of a human papillomavirus E6 / E7 mRNA detection kit are as follows:

[0056]

Embodiment 2

[0057] Embodiment two: the design of detection primer and probe

[0058] Firstly, the whole genome DNA sequences of 14 high-risk HPV types were downloaded from the NCBI gene bank in the United States to find the E6 / E7 gene sequences, and the ORF sequence of the internal reference gene β-Actin was downloaded and combined with the software Oligo 7.56 (Molecular Biology Insights, Inc. USA) for primers and Probe design. The design idea is: comprehensively consider the specificity and generality of the detection of 14 high-risk HPV E6 / E7 genes, and the general principles that should be followed in the design of primers and probes (such as Tm value, 3' end free energy, GC content, avoid appearance of internal structures and formation of dimers, etc.). After the designed primers and probes are synthesized, they are screened and verified by qPCR experiments. Finally, the present invention selects the optimal primers and probes. The nucleotide sequence is as follows: the nucleosides o...

Embodiment 3

[0067] Embodiment three: the preparation of positive quality control product and negative quality control product

[0068] 1. Preparation of positive quality control products and other types of pseudovirus particles

[0069] A commercially available commercial nucleic acid extraction kit was used to extract viral RNA from samples infected with various types of HPV, and perform PCR amplification (amplified sequence SEQ ID.46-SEQ ID.59). The amplified target band was recovered, ligated with the modified pET32-MS3his vector, ligated overnight at 4°C, the ligated product was transformed into BL21(DE3)pLysS competent cells, resistance screening, shaking, PCR identification For positive cases, the recombinant plasmid was extracted and verified by sequencing. Obtain the cloning bacteria solution containing the target fragment and induce expression with IPTG inducer, collect the induced bacteria solution and perform ultrasonic crushing to separate the supernatant and precipitate, and...

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Abstract

The invention relates to a kit for detecting high-risk human papillomavirus E6 / E7 mRNA, and discloses a detection method of the kit. The method comprises: 1, performing reverse transcription by usingrandom primers; and 2, detecting 14 kinds of high-risk HPV E6 / E7 mRNAs and an internal reference gene in a single PCR reaction. According to the present invention, with the primers and the probes designed for the 14 kinds of the high-risk HPVs, the high-risk HPV E6 / E7 mRNA can be specifically and rapidly detected, wherein the detection time is only 2.5 h; and the kit has characteristics of rapidness, high efficiency, high sensitivity, good specificity, real-time detection analysis and the like, provides the rapid and accurate molecular detection method for the general investigation of HPV andthe prevention and treatment of cervical cancer, has the greatly-reduced inspection cost, and further has important promotion and application value.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and in particular relates to a primer for detecting high-risk human papillomavirus E6 / E7 mRNA, a kit and a detection method thereof. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) belongs to papillary polyomavacuoviridae, is an epitheliophilic virus. It has a special tropism for the epidermis and mucosal squamous epithelium. It is a non-enveloped double-stranded circular DNA virus with high specificity and is widely distributed in humans and animals. The HPV genome consists of three gene regions, including the early region (Early Region, E region), the late region (Late Region, L region) and the non-coding region (Uncoding Region, UCR) or upstream regulatory region (URR). The E region consists of seven genes in sequence: E6, E7, E1, E2, E3, E4, and E5, which are involved in the replication of viral DNA, transcription, encoding viral proteins, and main...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/708C12Q2600/16
Inventor 任绪义潘彩霞吕江峰
Owner HANGZHOU DIAN BIOTECH CO LTD
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